Inhibition of complement mediated inflammatory response

ABSTRACT

A composition and methods for use thereof relating to polypeptides having the ability to act as an inhibitor of complement C5b-9 complex activity. The compositions contain an 18 kDa protein found on the surface of human erythrocytes, a 37 kDa protei 
     The U.S. Government has rights in this invention by virtue of certain government grants.

The U.S. Government has rights in this invention by virtue of certaingovernment grants.

BACKGROUND OF THE INVENTION

The present invention generally relates to compositions, and methods foruse thereof, effective in regulating inflammatory platelet andendothelial stimulatory and coagulopathic responses by modulating theactivity of the C5b-9 complex of the human plasma complement system.

The complement system is a complex interaction of plasma proteins andmembrane cofactors which act in a multi-step, multi-protein cascadesequence in conjunction with other immunological systems of the body toprovide immunity from intrusion of foreign cells. Complement proteinsrepresent up to about 10% of globulins in normal serum of man and othervertebrates.

The classic complement pathway involves an initial antibody recognitionof, and binding to, an antigenic site (SA) on a target cell. Thissurface bound antibody subsequently reacts with the first component ofcomplement, Clq, forming a Cl-antibody complex with Ca++, Clr, and Clswhich is proteolytically active. Cls cleaves C2 and C4 into activecomponents, C2a and C4a. The C4b,2a complex is an active protease calledC3 convertase, and acts to cleave C3 into C3a and C3b. C3b forms acomplex with C4b,2a to produce C4b,2a,3b, which cleaves C5 into C5a andC5b. C5b combines with C6. The C5b,6 complex combines with C7 to formthe ternary complex C5b,6,7. The C5b,6,7 complex binds C8 at the surfaceof the cell, which may develop functional membrane lesions and undergoslow lysis. Upon binding of C9 to the C8 molecules in the C5b,6,7,8complex, lysis of bacteria and other foreign cells is rapidlyaccelerated.

Recently, the C5b-9 proteins of the human plasma complement system havebeen implicated in non-lytic stimulatory responses from certain humanvascular and blood cells. The capacity of C5b-9 to modify membranepermeability and to selectively alter ion conductance is thought toelicit these non-lytic responses from human cells. In the case of humanblood platelets and vascular endothelium, assembly of the C5b-9 complexinitiates a transient and reversible depolarization of the plasmamembrane potential, a rise in cytosolic Ca2+, metabolic conversion ofarachidonate to thromboxane or prostacyclin, and the activation ofintracellular protein kinases. In addition, human platelets exposed toC5b-9 undergo shape changes, secretory fusion of intracellular storagegranules with plasma membrane, and the vesiculation of membranecomponents from the cell surface. Human endothelial cells exposed to thehuman C5b-9 proteins secrete high molecular weight multimers of theplatelet adhesion protein, von Willibrand Factor (vWF), and theintracellular granule membrane protein, GMP140, is translocated from theWeible-Palade body to the endothelial surface. High molecular weightmultimers of vWF have been implicated in the pathogenesis ofvaso-occlusive platelet adherence to endothelium and cell surface GMP140has been implicated in the adherence of inflammatory leukocytes toendothelium.

These effects of complement proteins C5b-9 on platelet and endothelialcells alter the normal regulation of the enzymes of the plasmacoagulation system at these cell surfaces. For example, the generationof platelet membrane microparticles by vesiculation results in theexposure of membrane binding sites for coagulation factor Va. Binding offactor Va to these membrane microparticle sites initiates assembly ofthe prothrombinase enzyme complex. This complex in turn acceleratescoagulation factor Xa activation of prothrombin to thrombin whichpromotes plasma clotting. Similarly, C5b-9 binding to the endothelialcell results in the exposure of plasma membrane receptors for theprothrombinase complex, thereby accelerating the generation of thrombinfrom prothrombin at the endothelial surface.

This interaction between components of the complement and coagulationsystems at the surface of blood platelets and endothelium can generateinflammatory and chemotactic peptides at sites of vascular thrombusformation and may contribute to the altered hemostasis associated withimmune disease states. In addition, immune reactions affecting bloodplatelets and endothelium can lead to platelet aggregation, thesecretion of proteolytic enzymes and vasoactive amines from plateletstorage granules, and increase adherence of platelets and leukocytes tothe endothelial lining of blood vessels.

It has been demonstrated that membrane-uptake of C3b and C5b-9 proteinscan occur spontaneously during incubation of platelets in citratedplasma. Complement activation can also occur during blood collection asa result of exposure to plastic surfaces supporting the C3-convertasereaction. While the implications of complement activation during bloodcollection and in vitro storage for transfusion have not been directlyaddressed it is, nevertheless, known that plasma levels of coagulationfactors V and VIII rapidly decline in stored platelet concentrates at arate considerably faster than their decay in cell-free plasma,suggesting consumptive loss. Further, platelet collection and storage isassociated with an increase in vesicular plasma membrane microparticles,a product of C5b-9 initiated platelet secretion. These physiological andenzymatic changes greatly reduce the potential shelf life of storedplatelets, particularly platelet-rich plasma concentrates used fortransfusions, which is generally only 72 hours at best. Furthermore,this interaction of activated C5b-9, platelets, and coagulation factorsin stored platelet concentrates will adversely affect the hemostaticeffectiveness of these units when infused.

In vitro human organ and tissue storage and survival of the transplantedgraft is also adversely affected by the spontaneous activation of thecomplement system, resulting in membrane insertion of the C5b-9 proteinsinto vascular endothelium. Activation of C5 to C5a and C5b has beenshown to be catalyzed by plastics and other synthetic membranes requiredto maintain perfusion of vascular beds during in vitro tissue and organstorage. In addition, membrane deposition of C5b-9 in vivo has beenimplicated in the acute rejection of transplanted tissue due to immuneactivation of the recipient's plasma complement system against theendothelial cells within the donor's organ.

Platelet and endothelial cell activation by C5b-9 also has ramificationsin autoimmune disorders and other disease states. The importance ofspontaneous complement activation and the resulting exposure ofplatelets and endothelium to activated C5b-9 to the evolution ofvaso-occlusive disease is underscored by consideration that a) leukocyteinfiltration of the subendothelium, which is known to occur in regionsof atheromatous degeneration and suggests localized generation of C5a atthe vessel wall, is potentially catalyzed by adherent platelets and b)local intravascular complement activation resulting in membranedeposition of C5b-9 complexes accompanies coronary vessel occlusion andmay affect the ultimate extent of myocardial damage associated withinfarction.

It is therefore an object of the present invention to provide a meansand method for the modulation and inhibition of complement C5b-9mediated platelet and endothelial cell activation in vivo and in vitro.

It is a further object of the present invention to provide a means andmethod for increasing the survival and therapeutic efficacy of plateletsand tissues or organs collected and stored in vitro.

It is a still further object of the present invention to provideprotection to transplanted organs or tissue and transfused blood cellsby prior in vitro incorporation of a membrane inhibitor of the C5b-9complex.

It is another object of the present invention to provide methods oftreatment for selected autoimmune disorders and other disease states.

SUMMARY OF THE INVENTION

A composition and methods for use thereof relating to polypeptideshaving the ability to act as an inhibitor of complement C5b-9 complexactivity. The compositions contain an 18 kDa protein found on thesurface of human erythrocytes, a 37 kDa protein found on the surface ofhuman platelets, a 37 kDa protein found on the surface of humanendothelial cells, active derivatives or fragments thereof which act toinhibit the activity of C5b-9, anti-idiotypic antibodies mimicking theaction of the inhibitor proteins or antibodies against C7 or C9 whichblock the formation of the C5b-9 complex.

The compositions can be used in vitro to inhibit C5b-9 relatedstimulatory responses of platelets and vascular endothelium of perfusedorgans and tissues, thereby preventing the C5b-9 initiated cell necrosisor stimulated secretion of proteolytic enzymes and the exposure of theprocoagulant membrane receptors during collection and in vitro storage.In one variation of this embodiment, the vascular endothelium of organsand tissues to be transplanted are treated with these compositions toprotect these cells from complement activation after transplantation. Inanother embodiment, immune disease states are treated by administeringan effective amount of a C5b-9 inhibitor to suppress C5b-9 mediatedplatelet activation in vivo.

Also disclosed are methods for the production of isolated polypeptidesthat are able to suppress complement C5b-9 mediated platelet andendothelial cell activation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of α-P18 on C9 incorporation into plateletmembrane C5b-9 complexes. Platelets were incubated with 100 μg α-P18/ml(solid bars) or 0 μg α-P18/ml (hatched bars) and the amount ofFITC-labeled anti-C9 neo-epitope antibody that bound to the platelets inthe presence of various concentrations of C8 (0, 0.015, 0.06, 0.25, and1 μg C8/10⁸ platelets) was measured by fluorescence.

FIGS. 2A and 2B show the effect of α-P18 on C5b-9-induced α-and densegranule secretion. Platelet α- (FIG. 2A) and dense (FIG. 2B) granulematerial secretion was measured after incubation of platelets witheither 0 μg α-P18/ml (---) or 100 μg α-P18/ml (-X--X-) in the presenceof various concentrations of C8 (0, 0.015, 0.06, 0.25, and 1 μg C8/10⁸platelets). The release of LDH is also shown by (Δ) for release in theabsence of α-P18 and by (□) in the presence of α-P18.

FIGS. 3A, 3B, and 3C show the platelet response as a function of α-P18Fab concentration, 0, 7.8, 31.3, and 125 μg α-P18/ml: α-granulesecretion as measured by FITC-S12 binding (FIG. 3A), GPIb-containingmicroparticles formation (FIG. 3B), and coagulation factor Va bindingsite exposure as measured by FITC-V237 binding (FIG. 3C). Solid barsdenote α-P18 treated platelets and hatched bars denote control C5b-9platelets.

FIG. 4 shows the effect of α-P18 on C5b-9 -induced expression of thePACI epitope in cell surface GPIIb-IIIa, as a function of C8concentration, 0, 0.015, 0.06, 0.25, and 1 μg C8/10⁸ platelets. Hatchedbars denote FITC-labeled anti-GPIIb-IIIa antibody binding to controlC5b-9 platelets and solid bars denote FITC-labeled anti-GPIIb-IIIaantibody binding to platelets pretreated with 100 μg/ml α-P18.

FIGS. 5A and 5B show the effect of α-P18 on expression of plateletprocoagulant activity, as a function of C8 concentration, 0, 0.015,0.06, 0.25, and 1 μg C8/10⁸ platelets. FIG. 5A shows the level of factorVa binding site measured using the FITC-labeled monoclonal antibodyV237, directed against the factor Va light chain, measured in thepresence of 2 μg/ml of the ligand. FIG. 5B represents measuredprothrombinase activity (thrombin units/min×10⁸ platelets). Both FIG. 5Aand 5B compare using control C5b-9 (hatched bars) or treated (100 μg/mlα-P18) (solid bars) platelets. Data for complement free controls arealso shown.

DETAILED DESCRIPTION OF THE INVENTION

37 kDa platelet and endothelial plasma membrane inhibitors of the C5b-9complex which normally serve to attenuate the procoagulant responses ofblood platelets and endothelium exposed to activated complement proteinshave been discovered by cross-reaction with a monospecific antibody(α-P18) raised against an 18 kDa erythrocyte membrane inhibitor of C5b-9which was identified by Sugita, et al., J. Biochem. (Japan),104:633-637, (1988). The existence of these proteins and the studiesdetailed below indicate that a deletion or inactivation of these cellsurface components would increase the risk of vascular thrombosis andlead to a decreased storage time for platelets and platelet rich plasma(PRP), and perfused organs and transplanted tissue. Accordingly, thesurvival and hemostatic efficacy of platelets, and the survival oforgans and tissue for transplant, which are collected and stored invitro, can be increased by addition of the C5b-9 inhibitor to thestorage buffer or perfusate. Autoimmune disorders and other diseasestates that involve C5b-9 mediated platelet activation, including lupus,rheumatoid arthritis, and other types of immuno-vasculitis, can also betreated by the intravascular administration an effective amount of theinhibitor to suppress C5b-9 activity, or a composition containing apolypeptide C5b-9 inhibitor to a patient requiring such treatment.Similar uses of the inhibitor may be applicable for cell culture inhuman blood derived culture media.

The conclusions as to the mechanisms by which the platelet boundinhibitor inhibits the C5b-9 inflammatory response is based on thefollowing. Addition of the purified 18 kDa protein, isolated from humanerythrocyte membranes, to other blood cells or endothelium serves toprotect these cells from both the cytolytic and cell-stimulatory effectsof the C5b-9 complement proteins. The function of this 18 kDa C5b-9inhibitory protein, when bound to platelet and endothelial cellsurfaces, was also probed by raising a neutralizing (blocking) antibody(α-P18) that abrogates the C5b-9 inhibitory function of the purifiedmolecule in vitro as well as the endogenous C5b-9 inhibitory factors,which may include the 18 kDa and 37 kDa proteins. When bound to theplatelet surface, the FAB of α-P18 increases C9 activation by membraneC5b-8, as monitored by exposure of a complex-dependent C9 neo-epitope.Although α-P18 causes little increase in the cytolysis of plateletstreated with C5b- 9 (as determined from the total release of lactatedehydrogenase of less than 5%), it markedly increases the cellstimulatory responses induced by these complement proteins, includingsecretion from platelet alpha and dense granules, conformationalactivation of cell surface GP IIb-IIIa, release of membranemicroparticles from the platelet surface, and exposure of new membranebinding sites for components of the prothrombinase enzyme complex. Priorincubation of C5b67 platelets with 100 μg/ml α-P18 (Fab) lowers byapproximately 10-fold the half-maximal concentration of C8 required toelicit each of these responses in the presence of excess C9. Incubationwith α-P18 (Fab) alone does not activate platelets, nor does incubationwith this antibody potentiate the stimulatory responses of plateletsexposed to other agonists.

As used herein in the compositions and methods for the prolongation ofplatelet and organ survival and enhancement of therapeutic efficacy orsuppression of complement mediated disorders, "C5b-9 inactivator" refersto the 37 kDa protein from platelets, the corresponding 37 kDa proteinon endothelial cells, the 18 kDa protein on erythrocyte membranes,peptide fragments thereof having C5b-9 inhibitory activity, andpreferably containing a membrane binding domain, whether isolated fromnaturally produced materials or recombinantly engineered sequences,monoclonal antibodies to C7 that block membrane binding of the C5b-9 ,monoclonal antibodies to C9 that block C9 polymerization and insertioninto the membrane, monoclonal antibodies that blocks C9 binding to C5b-9, and anti-idiotypic antibodies which inhibit the function of the cellsurface molecules in inhibiting C5b-9 activity, especially the Fabfragments of monoclonal antibodies having this activity. All molecularweights are determined by SDS-PAGE under non-reducing conditions. The 37kDa and 18 kDa proteins are species specific, i.e., only inhibitorproteins of human origin will inhibit human C5b-9 .

These studies are described in more detail below.

Experimental Procedures Materials:

Bovine serum albumin (globulin and fatty acid free), prostaglandin E1,apyrase, phorbol myristate acetate (PMA) and N-hydroxysuccinimide biotinester were obtained from Sigma Chemical Co., St. Louis, MO;(p-amidino-phenyl)methanesulfonyl fluoride (pAPMSF) was from Med Cal;fluorescein-5-isothiocyanate (isomer I) was from Molecular Probes;phycoerythrin-streptavidin conjugate was from Southern BiotechnologyAssociates (Birmingham, AL) and Spectrozyme TH was from AmericanDiagnostica (Greenwich, CT). IODO-GEN was from Pierce Chemical Co.;Na¹²⁵ from ICN Biochemicals, and [¹⁴ C]serotonin was from Du Pont-NewEngland Nuclear. Human complement proteins C5b6, C7, C8 and C9 werepurified and analyzed for functional activity according to methodspreviously described by Wiedmer and Sims, J. Biol. Chem. 260, 8014-8019(1985). Bovine factors Va, Xa, prothrombin, thrombin, and the lightchain of factor Va were gifts from Dr. Charles T. Esmon (OklahomaMedical Research Foundation). All other chemicals were of reagent oranalytical grade.

Solutions

Solution I: 145 mM NaCl, 4 mM KCl, 0.5 mM Mg Cl₂, 0.5 mM sodiumphosphate, 0.1% (w/v) glucose, 0.1% bovine serum albumin, 5 mM PIPES, pH6.8.

Solution II: 137 mM NaCl, 4 mM KCl, 0.5 mM MgCl₂ 0.5 mM sodiumphosphate, 0.1% glucose (w/v), 0.12% bovine serum albumin, 20 mM HEPES,pH 7.4.

Erythrocyte Membrane Protein Inhibitory for C5b-9

The 18 kDa human erythrocyte protein inhibitory for C5b-9 lysis wasisolated by modification of methods described by Sugita, et al. (1988).Additional purification was obtained by Mono-Q™ FPLC (Pharmacia). Whenincorporated into erythrocytes, this protein inhibited the hemolyticactivity of the purified human C5b-9 proteins, due to inhibition of C9activation by membrane C5b-8. When subjected to 12% polyacrylamideSDS-PAGE (non-reducing), all of the C5b-9 -inhibitory activity of thisprotein was found to elute from a gel slice corresponding to a singleprotein band at 18 kDa molecular weight.

In addition to classical protein purification using columnchromatography, an example of which is discussed above, polypeptideshaving inhibitory activity can also be affinity purified using inhibitorspecific antibodies. Antibodies, such as α-P18 disclosed below, whichbind the C5b-9 inhibitor polypeptide, are immobilized on chromatographicmatrix material by techniques well known to those skilled in the art.

Erythrocytes or platelets containing the C5b-9 inhibitor protein ontheir surface are isolated from whole blood by centrifugation anddetergent solubilized. The resultant solubilized crude extract is thenmixed with matrix immobilized antibody either in a batch process orchromatographically. The immobilized antibodies specifically bind C5b-9inhibitor polypeptides while the remainder of the crude extract iseasily removed by washing. The purified inhibitor is then eluted andcollected.

Alternatively, polypeptides having the ability to inhibit C5b-9 mediatedprocoagulant responses are produced recombinantly using methods andtechniques well known to those in the art. For example, human DNA isisolated and digested with restriction enzymes to create fragments ofappropriate size and with appropriate cohesive ends to be ligated intoany of the known and commercially available (e.g. Promega's lambda gtllvector system) expression vectors. Alternatively, the isolated DNA issheared and the appropriate linkers are ligated onto the resultingfragments which are then inserted into the expression vector of choice.

Vectors containing human DNA fragments are next transformed into theappropriate bacterial strain, normally a strain of E. coli that isincluded in the expression vector kit, to generate the DNA gene bank orlibrary. Plating out the vector containing bacteria of the library onappropriate media results in expression of the inserted human DNAfragment. The colonies are screened for the presence of DNA encoding andexpressing the C5b-9 inhibitory polypeptide using specific antibodiessuch as α-P18 disclosed below. Positive colonies are isolated and usedfor the large scale expression of recombinantly produced inhibitoryprotein.

In this fashion intact inhibitory protein can be made recombinantly aswell as modified polypeptides and functional fragments and derivativesthereof. Functional polypeptides possessing the ability to inhibit C5b-9can be produced by any of the above discussed method or by othertechniques commonly known to those of ordinary skill in the art. Theseisolated and purified polypeptides can be further mixed withpharmaceutically acceptable carriers to form compositions for use inprolonging cell storage or in treatment of immune disorders or diseases.

Antibodies

Murine monoclonal antibody S12, specific for the platelet α-granulemembrane glycoprotein, GMP-140, was a gift from Dr. Roger P. McEver(Oklahoma Medical Research Foundation, Oklahoma City). Murine monoclonalantibody APl, specific for membrane glycoprotein, GP Ib, was from Dr.Thomas J. Kunicki (Blood Center of Southeastern Wisconsin, Milwaukee).Murine monoclonal PACI, specific for the activated conformation of theGP IIb-IIIa complex, was from Dr. Sanford J. Shattil (University ofPennsylvania, Philadelphia). Murine monoclonal antibody MAC, specificfor a neo-epitope in C9 exposed upon its incorporation into membraneC5b-9 or SC5b-9 , was from Dr. John Tamerius (Cytotech Corp.). Murinemonoclonal antibody V237 recognizes an epitope in the light chain offactor Va (human or bovine) and binds to both factor V and factor Va.Murine monoclonal antibodies against the human C5b-9 proteins wereraised by immunization against the purified human proteins and thenscreened for inhibitory activity towards the cytolytic or cellactivating function of the C5b-9 complex. These monoclonal antibodies,or their Fab fragments, mimic the inhibitory function of the plasmamembrane C5b-9 inhibitor in that they raise the threshold amount ofactivated C5b-9 required to achieve either red cell lysis or plateletand endothelial cell activation.

Monospecific rabbit antibody against the purified human erythrocyte 18kDa protein (α-P18) was raised by repeated injection of the purified andisolated C5b-9 inhibitory polypeptide antigen isolated by themodification of the method of Sugita, et al. (1988). Immunized rabbitblood was collected and the IgG fraction was isolated by absorption toimmobilized staph protein A, followed by gel permeation chromatographyon Sephadex G200 (Pharmacia). Western blotting against detergentsolubilized extracts of human erythrocyte membrane proteins demonstratedthat this antibody was monospecific for a single protein of 18 kDa(non-reduced). Reactivity of this antibody was lost upon disulfidereduction of the antigen.

Fab fragments of IgG were prepared by 2 h digestion at 37° C. withimmobilized papain (Pierce). The resulting Fab fragments were purifiedto homogeneity by absorption against immobilized staph protein A and gelpermeation chromatograph (Sephadex™ G150; Pharmacia).

Fluorescence Labelino

For flow cytometry, all antibodies were conjugated with FITC, exceptantibody API, which was conjugated with N-hydroxysuccinimide biotinester as described previously. Dye-to-protein ratios ranged from 3-to-5.

Protein Concentrations

Concentrations of unlabeled proteins were estimated assuming thefollowing extinction coefficients (E^(1%) ₂₈₀): murine IgG (15), factorVa (15.1), factor Va light chain (18.7), factor Xa (12.4), prothrombin(15.5), C5b6 (10), C7 (9.9), C8 (15.1), and C9 (9.6). The concentrationsof FITC-labeled proteins were determined by dye binding assay (BioRad),using the respective unlabeled protein as standard. FITC concentrationwas determined assuming a molar extinction (492 nm) of 68,000.

Effect of α-P18 on Platelet Activity by C5b-9

Gel-filtered human platelets were prepared and collected into Solution Iat 1-2×10⁹ ml as described by Wiedmer and Sims, J. Biol. Chem. 260,8014-8019 (1985) or Wiedmer, et al., J. Biol. Chem. 262, 13674-13681(1986). To assemble membrane-bound C5b67 complexes, gel-filteredplatelets (10⁹ /ml) were incubated for 5 min at 37° C. with C5b6 (15μg/10⁸ platelets). The C5b67 platelets were then incubated with the Fabfragments of a α-P18 (0 to 125 μg/ml) for 10 min at room temperature.After dilution to 10⁸ platelets/ml in Solution II containing 2.5 mM CaCl₂, C8 (0 to 1 μg per 10⁸ cells) and C9 (0 or 4 μg per 10⁸ cells) wereadded, and the cells incubated at 37° C. without stirring for 10 min. Inall studies, comparison was made to identical matched-pair controls(platelets incubated in the absence of the C5b-9 proteins). In certainexperiments, comparison was also made to gel-filtered plateletsstimulated by incubation (10 min at 37° C. without stirring) with PMA(0-0.1 M) or thrombin (0.-0.5 U/ml).

Preparation of Platelets for Flow Cytometry

5×10⁸ C5b-9 treated or control platelets were incubated in the dark in atotal volume of 60 μl for 10 min at 23° C. in the presence of biotin-API(1 μg/ml) and one or more of the following fluorescein-conjugatedantibodies: FITC-α-P18 (Fab; 50 μg/ml), FITC-MAC (30 μg/ml), FITC-S12(10 μg/ml), or FITC-V237 (20 μg/ml). To measure membrane binding sitesfor factor Va, cells were first incubated for 10 min at 23° C. withfactor Va light chain (2 μg/ml) before addition of FITC-V237. Followingincubation with the labeled antibodies, phycoerythrin-streptavidin wasadded (5 μl of a 1:20 dilution), and the cells were incubated anadditional 10 min. Then 0.5 ml aliquots of Solution II were added andthe samples analyzed flow cytometry. All analyses were complete within30 min.

Flow Cytometry

Platelets were analyzed in a Becton Dickinson FACSCAN flow cytometerformatted for two-color analysis. The light scatter and fluorescencechannels were set at logarithmic gain. In order to resolveplatelet-derived microparticles from background light scatter,acquisition was gated so as to include only those particles distinctlypositive for biotin-APl (detected as phycoerythrin Fluorescence), usinga fluorescence lower-limit threshold on the 585 nm channel that excludedbackground scatter. Thus, only those cells and microparticles expressingthe platelet-specific membrane glycoprotein, GP Ib, were included foranalysis. Ten thousand phycoerythrin-positive particles from each samplewere analyzed for forward and right angle light scatter and for FITC andphycoerythrin fluorescence intensities. To measure FITC-α-P18 binding toerythrocytes, the threshold for acquisition was set on forward scatter.All fluorescence data were corrected for cell or microparticleautofluorescence (generally, ≦2 arbitrary fluorescence units perparticle). Where indicated, correction for non-specific binding was madeby incubation of cells with FITC-labeled antibody in the presence of a20-fold excess the unlabeled antibody (IgG or Fab).

Secretion and Cell Lysis Assays

Dense granule secretion was measured by the release of [¹⁴ C]serotonin.Alpha granule secretion was monitored by the surface expression ofGMP-140, detected by the binding of FITC-labeled monoclonal antibodyS12. Cell lysis was monitored by release of cytoplasmic lactic aciddehydrogenase.

Prothrombinase Assay

Platelet prothrombinase activity was measured by modification of methodspreviously described, using the chromogenic substrate Spectrozyme TH, byWiedmer, et al., Blood 68, 875-880 (1986). After activation, plateletswere diluted to a final concentration of 5×10⁶ per ml in Solution IIcontaining 1% albumin, 2.5 mM CaCl₂, 2 nM factor Va, and 2.7 μMprothrombin, and incubated at 37° C. Prothrombin conversion wasinitiated by addition of factor Xa to a final concentration of 2 nM. Thereaction was stopped at 0, 30, and 60 seconds by transfer of vol ofsample into 9 vol of ice cold buffer containing 1% albumin and 10 mMEDTA, and thrombin assayed as described by Wiedmer, et al., (1986).

Studies relating to the C5b-9 mediated activation of human culturedumbilical vein endothelial cells were performed as described by R.Hattori, et al., J. Biol. Chem. 264, 7768-7771

Results α-P18 Binds Specifically to the Platelet Plasma Membrane andIncreases the Incorporation of Activated C9 into Membrane c5b-9

The hemolytic activity of the terminal complement proteins appears to beregulated by two distinct proteins expressed on the surface of humanerythrocytes: "homologous restriction factor" (also referred to as"C8-binding protein") with an apparent molecular weight of 64 kDa, andan 18 kDa protein that does not exhibit C8-binding activity. Both ofthese membrane proteins have been shown to inhibit the cytolyticactivity of the human terminal complement proteins, apparently byaffecting the interaction of C9 with membrane bound C5b-8. Evidence hasbeen presented that homologous restriction factor is normally bound tothe cell surface by linkage to membrane phosphatidylinositol, and thatthis protein is deleted from the affected blood cells obtained frompatients with the disorder Paroxysmal Nocturnal Hemoglobinuria (PNH).

In order to determine whether the 18 kDa component of the erythrocytemembrane also plays a role in regulating the C5b-9 complex on theplatelet surface, an antibody (α-P18) that binds to the 18 kDaerythrocyte protein and abrogates its C5b-9 -inhibitory function wasproduced. In addition to binding erythrocytes, this antibody was foundto bind specifically to platelets, as demonstrated by the results inTable I.

                  TABLE I                                                         ______________________________________                                        Binding of α-P18 to Human Platelets and Red Blood Cells.sup.1                   ERYTHROCYTES                                                                              PLATELETS                                                 ______________________________________                                        FITC-Fab   86,100 ± 300                                                                            22,800 ± 200                                       FITc-IgG  415,100 ± 600                                                                            103,000 ± 600                                                              (mean ± s.d., n = 3)                               ______________________________________                                         .sup.1 Cell binding of FITClabeled α-P18 (IgG or Fab) was measured      and corrected for nonspecific binding as described in Materials and           Methods. Data are expressed as fluorescence (arbitrary units) per 10.sup.     cells.                                                                   

When bound to red cells, α-P18, or its Fab fragment, neutralized theC9-inhibitory activity of the 18 kDa membrane protein, and therebyincreased the lytic susceptibility of the cells to purified C5b-9 .Conversely, addition of the purified 18 kDa protein to blood cellsuspensions acted to inhibit cell lysis or cell activation by the C5b-9proteins. To test the effect of this antibody on platelets, gel-filteredhuman platelets were first exposed to the C5b67 proteins, and thenincubated with α-P18 (Fab fragments) before addition of C8 plus C9.

C5b67 platelets were incubated for 15 min at 23° C. with either 0(hatched bars) or 100 μg/ml (solid bars) α-P18 (Fab) before addition ofC9 (4 μg per 10⁸ cells) plus varying amounts of C8. After an additional10 min incubation at 37° C., samples were stained with FITC-labeledmonoclonal antibody against C9 neo-epitope (FITC-MAC) and processed forflow cytometry. The results are shown in FIG. 1. The ordinate denotestotal membrane-associated fluorescence (arbitrary units), corrected fornon-specific binding of FITC-MAC. Data for C9-free controls are alsoshown.

As shown by these data, incubation with α-P18 increased the amount ofactivated C9 incorporated into plasma membrane C5b-9 complexes,suggesting that this antibody also neutralizes a C5b-9 regulatoryfunction of the platelet membrane. In these studies, the activated formof C9 was detected by use of a fluorescently-labeled monoclonal antibody(FITC-MAC) that recognizes a neo-epitope in C9 that is expressed uponits incorporation into membrane C5b-9 .

Similar results were obtained using cultured umbilical cells.

Effect of α-P18 on C5b-9 Induced Secretion and Activation of CellSurface GPIIb-IIIa

The increased amount of activated C9 detected on the surface ofplatelets exposed to α-P18 suggested that this antibody might alsoaffect the susceptibility of these cells to the stimulatory or cytolyticeffects of the C5b-9 proteins. As shown in FIGS. 2 and 3, incubationwith α-P18 markedly potentiated the capacity of the C5b-9 proteins toinduce secretion from both α- and dense granules, but caused littleincrease in the platelet's susceptibility to lysis by these complementproteins.

α-Granule secretion (FIG. 2A) was monitored by the total binding ofmonoclonal antibody FITC-S12. Dense granule secretion (FIG. 2B) of thesame samples was monitored by the release of [14]C-serotonin. Cells wereexposed to either 0 or 100 μg/ml α-P18 (Fab) before C5b-9 assembly. Alsoshown is LDH release under these conditions: 0 μg/ml α-P18; 100 μg/mlα-P18. Data for complement-free controls (0vs. 100 μg/ml α-P18):α-granule secretion, 2.2% vs. 2.8%; dense granule secretion, 3.7% vs.3.6%; LDH release, 1.0% vs. 1.0%.

C5b67 platelets were incubated for 15 min at 23° C. with α-P18 (Fab;concentrations given on abscissa) before addition of C8 (1 μg per 10⁸platelets) plus C9 (4 μg per 10⁸ platelets). After additional incubationfor 10 min at 37° C., samples were processed for flow cytometry. FIG. 3Ashows α-granule secretion measured by FITC-S12 binding. FIG. 3B showsformation of GPIb-containing microparticles. FIG. 3C shows the exposureof factor Va binding sites measured by FITC-V237 binding. In each panel,hatched bars denote data for C5b-9 platelets and solid bars denote datafor C5b67 controls exposed to the identical concentrations of α-P18.

As demonstrated by FIG. 3A, this potentiation of C5b-9 stimulatedsecretion of platelet storage granules increased in a dose-dependentfashion with the concentration of the antibody. At these concentrations,α-P18 alone, or in combination with C5b-8, in the absence of added C9,did not directly stimulate platelet secretion nor did this antibodypotentiate the platelet's secretory response to stimulation by low dosethrombin or phorbol ester.

In addition to potentiating the platelet's secretory response to C5b-9 ,α-P18 also caused a marked increase in the C5b-9 -dependent binding ofmonoclonal antibody FITC-PACI to the platelet surface as shown in FIG.4. The PACl monoclonal antibody recognizes a conformational neo-epitopeexpressed by activated cell surface GP IIb-IIIa (the platelet'sfibrinogen receptor).

C5b-9 assembly and incubation with α-P18 was performed as described forthe data shown in FIG. 1, and conformational activation of membraneGPIIb-IIIa complexes probed by staining with FITC-PACl. Hatched barsdenote data for C5b-9 platelets (no α-P18); solid bars denote data forthe same platelets pre-treated with 100 μg/ml α-P18 (Fab) before C8 andC9 additions. Controls refer to complement-free platelets.

Effect of α-P18 on the Procoaqulant Activity of the C5b-9 Proteins

In addition to stimulating platelet and endothelial cell secretion, theC5b-9 proteins have been shown to initiate the release of small membranevesicles, approximately 100 nM diameter, also known as platelet factor3, from the cell surface that incorporate plasma membrane glycoproteinsIb, IIb, and IIIa, and the α-granule membrane derived glycoproteinGMP-140. The release of these membrane microparticles from the plateletsurface is directly coupled to the influx of Ca²⁺ into the plateletcytosol, and results in exposure of membrane receptors for factor Va andexpression of catalytic surface for the prothrombinase enzyme complex.As illustrated by FIG. 3B and flow cytometry showing the effect of α-P18on the release of membrane microparticles from platelets exposed toC5b-9 , prior treatment with α-P18 greatly increased the number ofmicroparticles released the platelet surface upon C9 binding to membraneC5b-8. In these studies, platelets and their derived membranemicroparticles were quantitated using biotin-AP1, as described by Sims,et al., J.Biol.Chem. (1988).

C5b67 platelets were incubated with either 0 or 100 μg/ml α-P18 (Fab)before addition of C8 and C9. After incubation for 10 min at 37° C., thecells were stained with monoclonal antibody APl (directed against GP Ib)and analyzed by flow cytometry. The red fluorescence threshold was setso that only GP Ib-positive particles were analyzed. Data forcomplement-free controls exposed to 0 or 100 μg/ml α-P18 was obtainedfor purposes of comparison. In these studies, the number ofmicroparticles detected (as percent of all GP-Ib-positive particlesanalyzed) was 9.7% and 9.2% for the controls, respectively, and 29% and74% with antibody. α-P18 alone did not induce vesiculation of theplatelet surface nor did this antibody increase the number ofmicroparticles detected when platelets were exposed to other agonists.The distribution of cell surface components between platelets and theshed microparticles is summarized in Table II.

                  TABLE II                                                        ______________________________________                                        Effect of α-P18 on Microparticle Formation.sup.1                                     FITC-MAC  FITC-V237                                                           Bound     Bound                                                            # Micro-         Micro-       Micro-                                α-  parti-   Plate-  parti-                                                                              Plate- parti-                                P18.sup.2 cles.sup.3                                                                             lets    cles  lets   cles                                  ______________________________________                                        C5b-9.sup.4                                                                          +      24,560   149,500                                                                             171,400                                                                             1,651,600                                                                            4,396,500                                  -      1,810    58,500                                                                              19,700                                                                              877,000                                                                              610,400                             C5b-8  +      790      0     0     160,000                                                                              35,900                                     -      790      0     0     166,900                                                                              28,300                              C5b67  +      790      0     0     149,900                                                                              30,900                                     -      790      9     4     159,200                                                                              30,700                              Control                                                                              +      770      0     0     171,500                                                                              28,100                                     -      760      --    --    174,900                                                                              27,300                              ______________________________________                                         Footnotes:                                                                    .sup.1 Distribution of FITClabeled antibodies between platelets and           plateletderived microparticles as discriminated by light scattering gates     Data expressed as total fluorescence in each gate (arbitrary fluorescence     units). All data corrected for autofluorescence and nonspecific binding o     labeled antibody.                                                             .sup.2 Platelets incubated in presence (+) or absence (-) of 100 μg/ml     α-P18 (Fab).                                                            .sup.3 Number of microparticles detected per 10.sup. 4 platelets.             .sup.4 C5b-9 (and intermediates) assembled as descsribed in Materials and     Methods, using 1 μg C8 per 10.sup.8 platelets per ml.                 

Inspection of these data reveals that the increase inmembrane-incorporated C9 due to α-P18 (plotted in FIG. 1) is largelyaccounted for by C5b-9 complexes bound to GP Ib-positive microparticles.Since all membrane C5b-8 is initially on the platelet surface (thevesiculation of microparticles from the plasma membrane initiated onlyafter C9 binds to its receptor, membrane C5b-8), these data suggest thatthe α-P18 mediated increase in plasma membrane-inserted C9 is ultimatelycompensated by the triggered vesiculation of assembled C5b-9 complexesfrom the platelet surface.

In addition to increasing C9 incorporation and microparticle formation,α-P18 caused a 4-to-5-fold increase in the number of factor Va bindingsites exposed upon C5b-9 binding to the platelet surface, as shown byFIGS. 3C and 5A. This increase in total factor Va binding sitesrepresented a 7-fold increase due to new sites exposed on microparticlesthat were released from the surface of a α-P18 treated platelets,combined with a two-fold increase in the number of factor Va bindingsites exposed on the cells themselves under these conditions, as shownby Table II. This increased exposure of factor Va binding sites due tothe presence of α-P18 resulted in a comparable increase in the C5b-9-induced expression of catalytic membrane surface for the prothrombinaseenzyme complex.

Platelets were incubated with α-P18 and the C5b-9 proteins underconditions described for the data shown in FIG. 1, and then analyzed forexposure of membrane sites for the prothrombinase enzyme complex, asshown in FIG. 5A. Factor Va binding sites were measured using amonoclonal antibody against factor Va light chain (FITC-V237), measuredin the presence of 2 μg/ml of the ligand, with the data representingtotal membrane sites for factor Va (platelet andmicroparticle-associated). FIG. 5B shows prothrombinase activitymeasured as described in Materials and Methods. Concentrations of α-P18(Fab) were either 0 or 100 μg/ml. Data for complement-free controls arealso shown.

In summary, these data suggest that the platelet and endothelial cellplasma membrane contain an inhibitor of the terminal complement C5b-9proteins that shares both functional and antigenic properties with the18 kDa protein recently identified in the human erythrocyte membrane. Inerythrocytes, this protein appears to serve a key role in restrictingthe cytolytic consequence of C5b-9 assembly. In addition to contributingto the normal resistance of human platelets and endothelial cells tolysis by complement, this membrane component appears to directlyattenuate the capacity of the C5b-9 proteins to induce procoagulantresponses and platelet and leukocyte adhesive endothelial responses.

In platelets exposed to α-P18, the half-maximal concentration of C8required for C5b-9 -induced secretion (FIG. 2), exposure of the PACIepitope in GPIIb-IIIa (FIG. 4), exposure of factor Va binding sites(FIG. 5A) and the expression of prothrombinase activity (FIG. 5B)decreased by more than 10-fold. This was accompanied by increasedbinding of activated C9 at each input of C8 (to pre-assembled plasmamembrane C5b67; FIG. 1).

Taken together, these data suggest that epitopes recognized by α-P18include functional domains of a membrane component that inhibitsformation of the C5b-9 complement pore, specifically by interfering withthe binding and/or activation of C9 by membrane bound C5b-8. Similarresults have been obtained in studies with erythrocytes and endothelialcells. The requirement for activated C9 (incorporated into membraneC5b-9 complexes) in the platelet responses observed in the presence ofthis antibody is underscored by the failure to detect significantplatelet activation when either C8 alone (in the absence of C9) wasadded to C5b67 platelets exposed to α-P18 (Table II), or, whensaturating amounts of C9 were added to these platelets in the absence ofadded C8 (FIGS. 2,4,5).

In addition to confirming that the effect of α-P18 is on the capacity ofthe platelet and endothelial cell plasma membrane to regulate assemblyof the C5b-9 pore (and not directly on the stimulatory state of the cellper se), these data exclude the possibility that the increasedactivation of the complement pore observed in the presence of thisantibody arose from trace contamination of the IgG (Fab) by eitherrabbit C8 or C9.

As shown in FIG. 2B, pre-treatment with α-P18 did slightly increase thelysis of platelets exposed to C5b-9 (as measured by release of LDH).Nevertheless, this effect on the platelet's susceptibility to cytolysisby C5b-9 was quite small, and total cell lysis never exceeded 5% underthe experimental conditions. This stands in contrast to the markedeffect of this antibody on the sensitivity of platelets to thecell-stimulatory effects of the C5b-9 proteins. Most notably, α-P18greatly increased both the number of membrane microparticles shed fromthe surface of C5b-9 treated platelets and the number of factor Vabinding sites exposed on these membrane surfaces (microparticle andplatelet). This capacity of the platelet to shed cell surface componentsis integrally related both to the observed inactivation of functionalC5b-9 pores, which restores the electrochemical integrity of the plasmamembrane, as well as to the exposure of membrane receptors for factorVa, which initiates the prothrombinase reaction.

The capacity of this blocking antibody to potentiate the C5b-9 -inducedexposure of factor Va binding sites, and thereby increase prothrombinaseactivity, suggests that a deletion or inactivation of the membraneepitopes recognized by this antibody would also potentiate theprocoagulant response of platelets exposed to low levels of complementactivation.

It is apparent from the above findings that inactivation of functionalC5b-9 inhibitor or a reduction in the platelet or endothelial cellmembrane concentration of C5b-9 inhibitor molecules would result inincreased platelet or endothelial cell activation. Conversely,administration of this inhibitor, or a polypeptide representing itsfunctional domain and possessing C5b-9 inhibitory activity, to blockplatelet or endothelial cell activation in a patient in need of suchtreatment, would thereby protect the patient from C5b-9 mediatedprocoagulant and prothrombotic responses.

In this context, it is important to note that affected red cellsobtained from patients with the acquired stem cell disorder ParoxysmalNocturnal Hemoglobinuria (PNH) have been shown to exhibit abnormalsensitivity to lysis by the C5b-9 proteins. This has been attributed tothe deletion of homologous restriction factor. Platelets obtained frompatients with PNH have been shown to be abnormally sensitive to fluidphase complement activation, and this disorder is characterized by anunusually high risk of venous thrombosis. The data with α-P18 suggest apossible mechanism by which loss of the C5b-9 inhibitory activity fromthe platelet plasma membrane would directly give rise to the thromboticepisodes that are associated with this disorder. This same finding isequally applicable to other types of complement mediated disorders,particularly in view of the discovery that the inhibitor is also foundon the surface of endothelial cells. As a result, administration of theinhibitor protein, whether purified from cells or expressed from cellsengineered using recombinant techniques, or portions of the peptidehaving the same measurable activity, can be administered to thesepatients to alleviate the severity of the disorder.

Treatment of patients with immune disorders and diseases such asimmunovasculitis, rheumatoid arthritis, scleroderma, disseminatedintravascular coagulation, lupus, paroxysmal nocturnal hemoglobinuria,thrombotic thrombolytic purpura, vascular occlusion, reocclusion aftersurgery, coronary thrombosis, and myocardial infarction, is accomplishedby administering an effective amount of a composition containing a C5b-9inactivator as defined above such that procoagulant processes aresuppressed. In the case of transfused blood cells or transplanted organsor tissue, the purified membrane inhibitor of C5b-9 , or thefunctionally equivalent polypeptide or antibody, is first coated on thecell surface before transplantation or transfusion into the recipient.The amount of composition that must be administered to a patient in needof such treatment will vary depending on the particular disorder ordisease and the severity of affliction. Treatment dosages will also varywith the individual patient depending upon response to treatment,genetic variability, and effect of co-administered drugs. In general,however, the compositions disclosed herein are administeredintravenously at a dosage of approximately nanograms of inhibitoryprotein or peptide per milliliter. Treatment can take the form of asingle administration of the composition or can be administeredperiodically or continuously over an extended period of time, asrequired.

For treatment of immune disorder or disease, the C5b-9 inactivator isadministered intravenously in a pharmaceutically acceptable carrier suchas saline or a physiologically acceptable buffer.

Isolated, functionally active polypeptides able to inhibit C5b-9 alsohave utility for increasing the hemostatic efficacy and extending the invitro storage time of blood and platelet preparations. There exists agreat need for prolonging the half-life, and therapeutic efficacy ofplatelets stored in vitro. Platelet-containing solutions, particularlyplatelet-rich plasma (PRP), are in tremendous demand medically fortransfusions. The current shelf life of platelet preparations isapproximately 72 hours. An increase in the useful lifetime of suchpreparations represents a significant advancement in the state of theart and answers a pressing human and medical need.

In the case of human organs and tissue for transplantation, the C5b-9inactivators would be added to the perfusate or storage medium toprotect the vascular lining cells from ongoing complement activationduring in vitro storage. Additionally, by coating these endothelialcells with a membrane-anchored C5b-9 inactivator, the organ or tissuewould be protected from the cytolytic and thrombotic effects arisingfrom complement activation initiated upon transplantation, therebycircumventing complement mediated acute rejection.

In the preferred embodiment, the C5b-9 inactivator is in combinationwith anticoagulant, such as ACD, CPD, heparin, or oxalate, such that theconcentration in the platelets or PRP is approximately nanogramsinactivator/ml. Similarly, for organ storage, the C5b-9 inactivator isin combination with perfusate or storage solutions, or culture medium,such that the concentration is approximately nanograms inactivator/ml.

It is apparent from the foregoing discussion that addition ofpolypeptides which act to inhibit the activity of C5b-9 towards humanplatelets and endothelium would reduce the incidence of C5b-9 mediatedprocoagulant and prothrombotic responses. Release of platelet granuleenzymes and factors result in clotting of platelets and generaldeterioration of the platelet preparation, limiting the shelf life ofsuch preparations. Thus, the addition of the disclosed compositionscontaining C5b-9 inhibitory polypeptides to platelet preparations willsuppress the spontaneous initiation of a procoagulant state and increasethe usable life of such preparations.

Compositions useful for extending the shelf life of plateletpreparations stored in vitro contain C5b-9 inhibitor in an amountsufficient to inhibit C5b-9 mediated platelet activation. Generally,these compositions will be added to platelet preparations, such asplatelet-rich plasma, such that the final concentration of inhibitorypolypeptide in the preparation is in the range of greater than 2Ki ofthe inactivator in the solution. For the 18 kDa protein and otherpolypeptides which incorporate a membrane binding domain, thetherapeutically effective dosage will be less than 1 μg inactivator/ml.Useful composition may also contain additional anticoagulant agents suchas oxalate, citrate, and heparin. The C5b-9 inhibitor containingcompositions can be added to whole blood as it is collected or toplatelet preparations after processing of the blood into isolatedplatelet concentrates.

Fluorescence-gated flow cytometry and monoclonal antibodies specific forselected plasma proteins (activated complement proteins and coagulationfactors) and for activated cell surface markers (GPIIb-IIIa, GMP140) canbe used to monitor the accumulation of cells surface C5b-9 , exposure ofmembrane receptors for coagulation factors fVa and fXa, activation ofplatelet secretion and platelet fibrinogen receptors, and the release ofplasma membrane derived platelet microparticles during in vitro cellstorage. These methods can also be used to monitor the secretion orremoval of C5b-9 inhibitory proteins from the platelet or endothelialcell surface. Additionally, the fluorescent membrane potentiometricdyes, such as the carbocyanine dyes, for example, diS-C3-5, can be usedto measure transmembrane electrochemical gradients indicative of cellviability and metabolic integrity. Use of both of these methods in bloodbanking will greatly decrease waste as well as therapeutic efficacy ofthe platelets which are determined not to be activated.

Modifications and variations of the present invention will becomeobvious to one skilled in the art in view of the description. Thesemodifications and variation are intended to fall within the scope of theappended claims.

We claim:
 1. A method for prevention of platelet and endothelial cellactivation and cytolysis by complement proteins comprising:adding amixture containing an effective amount to inhibit platelet orendothelial cell activation by C5b-9 of an 18 kDa C5b-9 inhibitoryprotein that is expressed on erythrocyte membranes, wherein themolecular weight of the inhibitory protein is determined by SDS-PAGEunder non-reducing conditions, and the inhibitory protein is of the samespecies of origin as the complement proteins to be inhibited; and acompound selected from the group consisting of anticoagulants, organperfusion media, and platelet and endothelial cell storage media; tocells selected from the group consisting of platelets and endothelialcells.
 2. The method of claim 1 further comprising treating endothelialcells prior to transplantation with an amount of said 18 kDa C5b-9inhibitory protein effective to prevent complement mediated activationand cytolysis after transplantation.
 3. The method of claim 1 furthercomprising mixing said 18 kda C5b-9 inhibitory protein withanticoagulant and collecting platelets into the mixture of C5b-9inhibitory protein and anticoagulant.
 4. The method of claim 1 whereinsaid 18 kda C5b-9 inhibitory protein is in a concentration of less thanapproximately one microgram inhibitory protein per milliliter of mixturewhen combined with the platelets or endothelial cells.
 5. The method ofclaim 1 wherein said 18 kda C5b-9 inhibitory protein is in aconcentration of greater than 2Ki of the inhibitory protein in themixture when combined with the platelets or endothelial cells.
 6. Acomposition for the prevention of platelet and endothelial cellactivation and cytolysis by complement proteins comprising:a mixturecontaining an effective amount to inhibit platelet or endothelial cellactivation by C5b-9 of an 18 kDa C5b-9 inhibitory protein that isexpressed on erythrocyte membranes, wherein the molecular weight of theinhibitory protein is determined by SDS-PAGE under non-reducingconditions, and the inhibitory protein is of the same species of originas the complement proteins to be inhibited; and a compound selected fromthe group consisting of anticoagulatns, organ perfusion media, andplatelet and endothelial cell storage media.